Understanding microRNA Biogenesis and Function

描述(由申请人提供)：microRNAs(MiRNAs)，~22nT长的单链RNAs，引导蛋白质复合体通过碱基配对阻止mRNAs的表达。第一个microRNA是在1993年发现的；第二个是在2000年发现的。目前，已在植物、动物和病毒中鉴定出3229个microRNAs。作为一类，miRNAs在协调基因表达变化方面的重要性可能与转录因子相媲美。我们的目标是了解miRNAs是如何合成、组装成功能复合体的，以及这些复合体是如何调节mRNA表达的。我们使用果蝇作为一个模型系统，因为它提供了强大的遗传和生化工具，而且因为miRNA途径在果蝇和人类之间是紧密保守的。我们在苍蝇身上学到的东西，我们在哺乳动物细胞提取物以及培养的人类和小鼠细胞系中进行测试。我们的目标是确定这些过程在苍蝇和哺乳动物中的哪些地方是保守的，以及它们在哪里有所不同，从而了解动物miRNA途径的共同逻辑以及哺乳动物进化的独特功能。前miRNAs是miRNAs的直接前体，是-65个核苷酸长的RNA茎环结构；前miRNA的茎是不完善的，G：U摆动对、错配和内部环中断了茎大约三个螺旋转弯的长度。我们将使用定量生化和分子工具来鉴定从前miRNAs产生miRNA所需的蛋白质和蛋白质复合体，并确定这些蛋白质如何提高前miRNA处理的准确性和效率。DICER是一种将前miRNAs转化为miRNAs的酶，需要双链RNA结合蛋白伙伴来催化miRNA成熟。对于所有的Pre-miRNA序列和结构，单个Dier蛋白伴侣是否足够，或者不同的双链RNA结合蛋白是否可以作为不同类别的Pre-miRNAs的DICER伙伴？一些miRNAs位于前miRNA茎的5‘臂；另一些位于3’臂。前miRNA的哪些序列和热力学特征确保从前miRNA茎的正确手臂产生正确的miRNA？MiRNAs在蛋白质-RNA复合体中发挥作用，蛋白质-RNA复合体的核心包含ArgAerte蛋白质家族的一个成员。苍蝇有五种不同的Argavite蛋白质；人类至少有七种。MiRNAs是如何--以及为什么--在不同的ArgAerte蛋白之间分配的？是什么决定了miRNA与哪种ArgAerte蛋白有关？是否含有相同的miRNA，但含有不同的ArgAerte蛋白的复合体，功能不同，每个都专门针对不同类型的mRNA靶标？我们试图了解miRNAs在苍蝇和人类中的生物学功能。为什么在miRNA生产中只受到轻微损害的果蝇会年轻死亡？抵抗环境胁迫是否需要miRNAs？最后，为了提供对miRNA途径的系统水平的看法，我们将开发新的实验工具来确定miRNA调节的信使核糖核酸种类以及通过哪个信使蛋白质调节。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs), ~22 nt long, single-stranded RNAs, guide protein complexes to block expression of mRNAs to which they bind by base pairing. The first microRNA was discovered in 1993; the second, in 2000. Currently, 3,229 microRNAs have been identified in plants, animals and viruses. As a class, miRNAs may rival transcription factors for their importance in orchestrating changes in gene expression. Our goal is to understand how miRNAs are made, assembled into functional complexes, and how these complexes regulate mRNA expression. We use Drosophila as a model system, because it offers powerful genetic and biochemical tools and because the miRNA pathway is closely conserved between flies and humans. What we learn in flies, we test in mammalian cell extracts and in cultured human and mouse cell lines. Our goal is to identify where these processes are conserved and where they diverge between flies and mammals, so as to understand the common logic of the miRNA pathway in animals and the unique features that have evolved in mammals. Pre-miRNAs, the immediate precursors of miRNAs, are -65 nt long RNA stem loop structures; the stems of pre-miRNA are imperfect, with G:U wobble pairs, mismatches, and internal loops interrupting a stem approximately three helical turns long. We will use quantitative biochemical and molecular tools to identify the proteins and protein complexes required to produce miRNA from pre-miRNAs, and to determine how these proteins enhance the accuracy and efficiency of pre-miRNA processing. Dicer, the enzyme that converts pre-miRNAs to miRNAs, requires a double-stranded RNA-binding protein partner to catalyze miRNA maturation. Does a single Dicer protein partner suffice for all pre-miRNA sequences and structures, or do different double-stranded RNA-binding proteins function as Dicer partners for distinct classes of pre- miRNAs? Some miRNAs reside in the 5' arm of the pre-miRNA stem; others, in the 3' arm. What sequence and thermodynamic features of the pre-miRNA ensure that the right miRNA is produced from the correct arm of the pre-miRNA stem? miRNAs function in protein-RNA complexes containing at their core a member of the Argonaute family of proteins. Flies have five different Argonaute proteins; humans have at least seven. How-and why-are miRNAs partitioned among different Argonaute proteins? What determines with which Argonaute protein a miRNA associates? Are complexes containing the same miRNA, but a different Argonaute protein, functionally distinct, each specialized for a different type of mRNA target? We seek to understand the biological functions of miRNAs in flies and humans. Why do flies only modestly impaired in miRNA production die young? Are miRNAs required for resistance to environmental stress? Finally, to provide a systems-level view of the miRNA pathway, we will develop new experimental tools to identify the mRNA species a miRNA regulates and through which Argonaute protein.